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High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae

DOI: 10.1073/pnas.0704546104 DOI Help

Authors: Simon Newstead (Division of Molecular Bioscience, Imperial College, U.K.) , Hyun Kim (Department of Biochemistry and Biophysics, Stockholm University, Sweden.) , Gunnar Von Heijne (Department of Biochemistry and Biophysics, Stockholm University, Sweden.) , So Iwata (Diamond Light Source) , David Drew (Division of Molecular Bioscience, Imperial College, U.K.)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences , VOL 104 (35) , PAGES 13936 - 13941

State: Published (Approved)
Published: August 2007

Abstract: Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Based on the analysis of 43 eukaryotic membrane proteins, we present a cost-effective high-throughput approach for rapidly screening membrane proteins that can be overproduced to levels of >1 mg per liter in Saccharomyces cerevisiae. We find that 70% of the well expressed membrane proteins tested in this system are stable, targeted to the correct organelle, and monodisperse in either Fos-choline 12 (FC-12) or n-dodecyl-?-d-maltoside. We illustrate the advantage of such an approach, with the purification of monodisperse human and yeast nucleotide-sugar transporters to unprecedented levels. We estimate that our approach should be able to provide milligram quantities for at least one-quarter of all membrane proteins from both yeast and higher eukaryotic organisms.

Journal Keywords: Gfp-Based Fusion Technology; Structural Genomics

Subject Areas: Biology and Bio-materials


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